Isolation And Identification Of Salmonella From Poultry In Eastern China During 2017 To 2018 And Establishment Of Indirect ELISA Method Basing On Lipopolysaccharide From Salmonella Pullorum

Salmonella is a zoonosis pathogen,which causes foodborne human gastroenteritis and poses a serious threat to the poultry industry,and avian Salmonella is one of important pathogens in Salmonella.Pullorum caused by host adapted S.pullorum is an acute or chronic infectious disease that can infect chickens with various ages through both horizontal and vertical transmission,and results in serious economic loss.The traditional detection methods of S.pullorum are the glass plate agglutination assay for anitibody and PCR assay for antigen.Although the glass plate agglutination test is simple and rapid,it is greatly affected by subjective judgment.Meanwhile,multiplex PCR detection technology has good specificity,while the cost of instruments and reagents is high.Therefore,it is necessary to establish a method with the characteristics of simple,rapid,sensitivity,specificity,and cheap for detecting S.pullorum.The O antigen polysaccharide chain of Lipopolysaccharides(LPS)with characteristic of highly variable specifically determines the serotype of bacteria,therefore LPS has good immunogenicity and antigenicity.The dectection method basing on LPS may determine specifically the antibody agsinst S.pullorum,which is important for quarantine and clean-up of S.pullorum.In this study,the suspected samples infected by Salmonella were collected in Eastern China during 2017 to 2018,Salmonella strains was isolated and the serotypes of the strains were identified by multiplex PCR and serological methods.Furthermore,bacterial sensitivity to 22 common antibiotics was determined.Finally,an indirect ELISA method was established to detect S.pullorum with the purified LPS from Salmonella S6702 and S44 strains.1.Isolation,Identification,and Drug Resistance of avian Salmonella in Eastern China during 2017-2018During 2017 to 2018,71 strains of Salmonella were identified by multiplex PCR and serological methods from suspected samples infected by salmonella in Eastern China,including 33 strains of S.typhimurium,25 strains of S.pullorum,10 strains of S.enteritidis,2 strains of S.Newport,and 1 strain of S.Derby.The drug resistance assay of these strains was carried out,and the results showed that the most of Salmonella isolates were resistance to ?-lactams and macrolides antibiotics.The multi-resistance rate of 71 strains was 92.96%,suggesting that the multi-resistance of Salmonella isolates in Eastern China during 2017-2018 was very serious.2.Extraction and identification of LPS from S.pullorumThe LPS of S.pullorum was extracted by an improved hot phenol-water method.The yield of LPS from Salmonella S6702 and S44 were 4.3% and 3.6%,respectively.The nucleic acid ratios in the purified LPS of the two strains were 0.011% and 0.011%,respectively;the protein ratios were 0.39% and 0.49%,respectively;the polysaccharides ratios were 18.3% and 16.5%,respectively.The SDS-PAGE and silver staining result showed a ladder type pattern of LPS,suggesting a smooth type of LPS,which meets the requirements for the subsequent studies.3.Establishmen of LPS-based indirect ELISA for detection the antibody against S.pullorumAn indirect ELISA method was established basing on the extracted LPS.The optimal reaction condition of the indirect ELISA was determined.The optimal concentration of coating antigen was 1.25 ?g/mL,the optimal dilution of the serum sample was 1:800,the blocking solution was 5%goat serum,the optimal dilution of the sheep anti-chicken enzyme-labeled antibody was 1:16000,the optimal reaction time of serum reaction and chromogenic liquid was 60 min and 15 min,respectively.The threshold of the indirect ELISA basing on S6702 LPS between negative samples and positive samples was 0.269.The positive serums of chickens infected with common pathogens such as Proteus,Providencia,Pasteurlla,Escherichia coli,Pseudomonas aeruginosa,and Salmonella typhimurium were all negative when detected by the established indirect ELISA,indicating that the detection method had good specificity.The titer of the positive serum detected by glass plate agglutination test was 1:64,while the titer detected by the ELISA was 1:12800,indicating that the ELISA is more sensitive.Moreover,the results of broad-spectrum,and reproducibility showed that the indirect ELISA had broad-spectrum with antisera against different S.pullorum isolates and good repeatability.After detection of 100 clinical sera,the coincidence rate between the indirect ELISA method and the glass plate agglutination assay was 89%,and the coincidence rate between the indirect ELISA method and imported ELISA kit was 93%.In summary,the multi-resistance of Salmonella in Eastern China during 2017 to 2018 is serious,and the LPS-based indirect ELISA method for S.pullorum has high specificity,good stability.Therefore,the indirect ELISA method is suitable for detecting S.pullorum in poultry farms and can provide technical support for the clear-up and elimination of pullorum disease.